This invention relates to infectious, attenuated viruses useful as vaccines against diseases caused by flaviviruses.
Several members of the flavivirus family pose current or potential threats to global public health. For example, Japanese encephalitis is a significant public health problem involving millions of at risk individuals in the Far East. Dengue virus, with an estimated annual incidence of 100 million cases of primary dengue fever and over 450,000 cases of dengue hemorrhagic fever worldwide, has emerged as the single most important arthropod-transmitted human disease.
Other flaviviruses continue to cause endemic diseases of variable nature and have the potential to emerge into new areas as a result of changes in climate, vector populations, and environmental disturbances caused by human activity. These flaviviruses include, for example, St. Louis encephalitis virus, which causes sporadic, but serious, acute disease in the midwest, southeast, and western United States; West Nile virus, which causes febrile illness, occasionally complicated by acute encephalitis, and is widely distributed throughout Africa, the Middle East, the former Soviet Union, and parts of Europe; Murray Valley encephalitis virus, which causes endemic nervous system disease in Australia; and Tick-borne encephalitis virus, which is distributed throughout the former Soviet Union and eastern Europe, where its Ixodes tick vector is prevalent and responsible for a serious form of encephalitis in those regions.
Hepatitis C virus (HCV) is another member of the flavivirus family, with a genome organization and replication strategy that are similar, but not identical, to those of the flaviviruses mentioned above. HCV is transmitted mostly by parenteral exposure and congenital infection, is associated with chronic hepatitis that can progress to cirrhosis and hepatocellular carcinoma, and is a leading cause of liver disease requiring orthotopic transplantation in the United States.
The Flaviviridae family is distinct from the alphaviruses (e.g., WEE, VEE, EEE, SFV, etc.) and currently contains three genera, the flaviviruses, the pestiviruses, and the hepatitis C viruses. Fully processed mature virions of flaviviruses contain three structural proteins, envelope (E), capsid (C), and membrane (M), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Immature flavivirions found in infected cells contain pre-membrane (prM) protein, which is the precursor to the M protein.
After binding of virions to host cell receptors, the E protein undergoes an irreversible conformational change upon exposure to the acidic pH of endosomes, causing fusion between the envelope bilayers of the virions and endocytic vesicles, thus releasing the viral genome into the host cytosol. PrM-containing tick-borne encephalitis (TBE) viruses are fusion-incompetent, indicating that proteolytic processing of prM is necessary for the generation of fusion-competent and fully infectious virions (Guirakhoo et al., J. Gen. Virol. 72(Pt. 2):333-338, 1991). Using ammonium chloride late in the virus replication cycle, prM-containing Murray Valley encephalitis (MVE) viruses were produced and shown to be fusion incompetent. By using sequence-specific peptides and monoclonal antibodies, it was demonstrated that prM interacts with amino acids 200-327 of the E protein. This interaction is necessary to protect the E protein from the irreversible conformational changes caused by maturation in the acidic vesicles of the exocytic pathway (Guirakhoo et al., Virology 191:921-931, 1992).
The cleavage of prM to M protein occurs shortly before release of virions by a furin-like cellular protease (Stadler et al., J. Virol. 71:8475-8481, 1997), which is necessary to activate hemagglutinating activity, fusogenic activity, and infectivity of virions. The M protein is cleaved from its precursor protein (prM) after the consensus sequence R-X-R/K-R (X is variable), and incorporated into the virus lipid envelope together with the E protein.
Cleavage sequences have been conserved not only within flaviviruses, but also within proteins of other, unrelated viruses, such as PE2 of murine coronaviruses, PE2 of alphaviruses, HA of influenza viruses, and p 160 of retroviruses. Cleavage of the precursor protein is essential for virus infectivity, but not particle formation. It was shown that, in case of a TBE-dengue 4 chimera, a change in the prM cleavage site resulted in decreased neurovirulence of this chimera (Pletnev et al., J. Virol. 67:4956-4963, 1993), consistent with the previous observation that efficient processing of the prM is necessary for full infectivity (Guirakhoo et al., 1991, supra; Guirakhoo et al., 1992, supra; Heinz et al., Virology 198:109-117, 1994). Antibodies to prM protein can mediate protective immunity, apparently due to neutralization of released virions that contain some uncleaved prM. The proteolytic cleavage site of the PE2 of VEE (4 amino acids) was deleted by site-directed mutagenesis of the infectious clone (Smith et al., ASTMH meeting, Dec. 7-11, 1997). Deletion mutants replicated with high efficiency and PE2 proteins were incorporated into particles. This mutant was evaluated in lethal mouse and hamster models and shown to be attenuated; in non-human primates it caused 100% seroconversion and protected all immunized monkeys from a lethal challenge.
The invention features chimeric, live, infectious, attenuated viruses that are each composed of:
(a) a first yellow fever virus (e.g., strain 17D), representing a live, attenuated vaccine virus, in which the nucleotide sequence encoding the prM-E protein is either deleted, truncated, or mutated so that the functional prM-E protein of the first flavivirus is not expressed, and
(b) integrated into the genome of the first flavivirus, a nucleotide sequence encoding the viral envelope (prM-E) protein of a second, different flavivirus, so that the prM-E protein of the second flavivirus is expressed from the altered genome of the first flavivirus.
The chimeric virus is thus composed of the genes and gene products responsible for intracellular replication belonging to the first flavivirus and the genes and gene products of the envelope of the second flavivirus. Since the viral envelope contains antigenic determinants responsible for inducing neutralizing antibodies, the result of infection with the chimeric virus is that such antibodies are generated against the second flavivirus.
A preferred live virus for use as the first yellow fever virus in the chimeric viruses of the invention is YF 17D, which has been used for human immunization for over 50 years. YF 17D vaccine is described in a number of publications, including publications by Smithburn et al. (xe2x80x9cYellow Fever Vaccination,xe2x80x9d World Health Org., p. 238, 1956), and Freestone (in Plotkin et al., (Eds.), Vaccines, 2nd edition, W. B. Saunders, Philadelphia, 1995). In addition, the yellow fever virus has been studied at the genetic level (Rice et al., Science 229:726-733, 1985) and information correlating genotype and phenotype has been established (Marchevsky et al., Am. J. Trop. Med. Hyg. 52:75-80, 1995). Specific examples of yellow fever substrains that can be used in the invention include, for example, YF 17DD (GenBank Accession No. U 17066), YF 17D-213 (GenBank Accession No. U17067), YF 17D-204 France (X15067, X15062), and YF-17D-204, 234 US (Rice et al., Science 229:726-733, 1985; Rice et al., New Biologist 1:285-296, 1989; C 03700, K 02749). Yellow Fever virus strains are also described by Galler et al., Vaccine 16 (9/10):1024-1028, 1998.
Preferred flaviviruses for use as the second flavivirus in the chimeric viruses of the invention, and thus sources of immunizing antigen, include Japanese Encephalitis (JE, e.g., JE SA14-14-2), Dengue (DEN, e.g., any of Dengue types 1-4; for example, Dengue-2 strain PUO-218) (Gruenberg et al., J. Gen. Virol. 67:1391-1398, 1988) (sequence appendix 1; SEQ ID NO:50; nucleotide sequence of Dengue-2 insert; Pr-M: nucleotides 1-273; M: nucleotides 274-498; E: nucleotides 499-1983) (sequence appendix 1; SEQ ID NO:51; amino acid sequence of Dengue-2 insert; Pr-M: amino acids 1-91; M: amino acids 92-166; E: amino acids 167-661), Murray Valley Encephalitis (MVE), St. Louis Encephalitis (SLE), West Nile (WN), Tick-borne Encephalitis (TBE) (i.e., Central European Encephalitis (CEE) and Russian Spring-Summer Encephalitis (RSSE) viruses), and Hepatitis C (HCV) viruses. Additional flaviviruses for use as the second flavivirus include Kunjin virus, Powassan virus, Kyasanur Forest Disease virus, and Omsk Hemorrhagic Fever virus. As is discussed further below, the second flavivirus sequences can be provided from two different second flaviviruses, such as two Dengue strains.
It is preferable to use attenuated inserts, for example, in the case of inserts from neurotropic viruses, such as JE, MVE, SLE, CEE, and RSSE. In the case of non-neurotropic viruses, such as dengue viruses, it may be preferable to use unmodified inserts, from unattenuated strains. Maintenance of native sequences in such inserts can lead to enhanced immunogenicity of the proteins encoded by the inserts, leading to a more effective vaccine.
In a preferred chimeric virus of the invention, the prM-E protein coding sequence of the second flavivirus is substituted for the prM-E protein coding sequence of the live yellow fever virus. Also, as is described further below, the prM portion of the protein can contain a mutation or mutations that prevent cleavage to generate mature membrane protein. Finally, as is discussed in detail below, the chimeric viruses of the invention include the prM signal of yellow fever virus.
Also included in the invention are methods of preventing or treating flavivirus infection in a mammal, such as a human, by administering a chimeric flavivirus of the invention to the mammal; use of the chimeric flaviviruses of the invention in the preparation of medicaments for preventing or treating flavivirus infection; nucleic acid molecules encoding the chimeric flaviviruses of the invention; and methods of manufacturing the chimeric flaviviruses of the invention.
The invention provides several advantages. For example, because they are live and replicating, the chimeric viruses of the invention can be used to produce long-lasting protective immunity. Also, because the viruses have the replication genes of an attenuated virus (e.g., Yellow Fever 17D), the resulting chimeric virus is attenuated to a degree that renders it safe for use in humans.